Identifying the Choline-Cation Tyrosine-PI Interactions of an Amphitropic Protein

Abstract

Transient binding of amphitropic proteins to membranes can be mediated by phospholipid cation / tyrosine π interactions. Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (BtPI-PLC) is a secreted virulence factor that targets GPI-anchored proteins in the outer surface of eukaryotic plasma membranes, a bilayer rich in choline-containing lipids. BtPI-PLC has a plethora of tyrosine residues around the surface of the α/β-barrel, and molecular dynamics (MD) simulations suggest that choline headgroups and these Tyr residues form short-lived cation-π complexes. To investigate these interactions, BtPI-PLC was site-specifically spin-labeled and high resolution field cycling 31P NMR relaxometry was used to quantify the effect of the spin-labeled BtPI-PLC variants on phosphatidylcholine (PC) and phosphatidylmethanol (PMe, used as the surrogate substrate) in small vesicles. The paramagnetic relaxation enhancement at very low fields (<0.04 T) confirmed the existence of two moderately long-lived sites for PC binding - one near the active site and the other on the barrel rim quite removed from the active site. The distances extracted are consistent with the two major sites of PC binding suggested by the simulations. That the discrete phospholipid sites detected by NMR represent cation-π interactions is shown by measuring the binding affinity for BtPI-PLC variants in which the unnatural amino acid 3,5-difluorotyrosine has been site-specifically substituted for Tyr, thus reducing the strength of cation-π interactions. These binding studies coupled with changes in enzyme activity provide a detailed and quantitative picture of how PC interactions with this PI-PLC influence its transient binding and cleavage of PI in membranes.